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A Steric-Inhibition Model for Regulation of Nucleotide Exchange Via the Dock180 Family of Gefs

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A Steric-Inhibition Model for Regulation of Nucleotide Exchange Via the Dock180 Family of Gefs CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Current Biology, Vol. 15, 371–377, February 22, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.cub.2005.01.050 A Steric-Inhibition Model for Regulation of Nucleotide Exchange via the Dock180 Family of GEFs Mingjian Lu,1 Jason M. Kinchen,3 Kent L. Rossman,4 and cell migration [1, 11]. These include both the cata- Cynthia Grimsley,1,2 Matthew Hall,5 John Sondek,4 lytic Docker domain that is located within the C-terminal Michael O. Hengartner,3 Vijay Yajnik,5 half of Dock180 and mediates nucleotide exchange on and Kodi S. Ravichandran1,* Rac and an approximately 350 amino acid N-terminal 1Beirne Carter Center for Immunology Research region that contains an SH3 domain. To better define Department of Microbiology and the molecular features of this N-terminal region of 2 Department of Pharmacology Dock180, we generated various Dock180 mutants (Fig- University of Virginia ure S1 in the Supplemental Data available with this arti- Charlottesville, Virginia 22908 cle online). Biochemical characterization of these mu- 3 Institute of Molecular Biology tants revealed two ELMO-interacting sites within this University of Zurich region. One interaction is mediated by the binding of 8057 Zurich, Switzerland the Dock180 SH3 domain to the PxxP motif of ELMO 4 Department of Pharmacology (Figure 1A). In addition, a second region adjacent to the University of North Carolina SH3 domain also binds ELMO and was disrupted by a Chapel Hill, North Carolina 27599 G171E mutation in Dock180 (Figure 1B; see below for 5 Gastrointestinal Unit description of G171E). It is noteworthy that these two Massachusetts General Hospital types of interaction are independent of each other. Harvard Medical School While characterizing these Dock180 mutants, we un- Boston, Massachusetts 02114 expectedly observed that the SH3-domain-deleted Dock180 was more active in a phagocytosis assay than wild-type Dock180 (Figure 1C, lanes 6 and 8), suggesting Summary that the SH3 domain of Dock180 might play a basal inhibitory role. One possible mechanism for such SH3- CDM (CED-5, Dock180, Myoblast city) family members mediated negative regulation is through binding to an- have been recently identified as novel, evolutionarily other region (or other regions) of Dock180. Consistent conserved guanine nucleotide exchange factors (GEFs) with this notion, the isolated SH3 domain (Dock1-83) or for Rho-family GTPases [1–7]. They regulate multiple a slightly larger N-terminal fragment of Dock180 (Dock1- processes, including embryonic development, cell mi- 161), coprecipitated with Dock (⌬SH3) (Figure 1D and gration, apoptotic-cell engulfment, tumor invasion, data not shown). No interaction was detectable between and HIV-1 infection, in diverse model systems [4, 6, the SH3 domain and the C-terminal proline-rich tail of 8–16]. However, the mechanism(s) of regulation of Dock180 (data not shown). When other regions of Dock180 CDM proteins has not been well understood. Here, our were examined, the Dock1–161 fragment or the isolated studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 SH3 domain readily bound the catalytic Docker domain family of GEFs. At basal state, the N-terminal SH3 (Figure 1E, lane 4; Figure S2) Intriguingly, the SH3: domain of Dock180 binds to the distant catalytic Docker interaction does not seem to be a typical Docker domain and negatively regulates the function SH3:PxxP binding; first, the W45A mutation within the of Dock180. Further studies revealed that the SH3: Dock180 SH3 domain, which abolished the domain’s Docker interaction sterically blocks Rac access to the binding to the PxxP motif of ELMO (Figure 1A), had no Docker domain. Interestingly, ELMO binding to the effect on binding to the Docker domain (Figure 1E, lane SH3 domain of Dock180 disrupted the SH3:Docker in- 5); second, disrupting the single PxxP motif within the teraction, facilitated Rac access to the Docker domain, Docker domain did not affect the Docker:SH3 interaction and contributed to the GEF activity of the Dock180/ (Figure 1E, lane 6). ELMO complex. Additional genetic rescue studies in We then aligned the amino acid sequences from the C. elegans suggested that the regulation of the SH3 domains of CDM family members and compared Docker-domain-mediated GEF activity by the SH3 do- the consensus sequence with those from other SH3 main and its adjoining region is evolutionarily conserved. domains. We identified a conserved isoleucine residue This steric-inhibition model may be a general mecha- (I32 of Dock180) within CDM family members; in most nism for regulating multiple SH3-domain-containing other SH3 domains, this residue is a lysine or glutamate Dock180 family members and may have implications (Figure S3). Of note, this I32 residue resides in the ligand for a variety of biological processes. binding RT loop of the SH3 domains. Mutating this iso- leucine residue to lysine (I32K) completely abrogated Results and Discussion the SH3:Docker interaction (Figure 1F, lane 3). We then tested whether the I32K mutation introduced An Inhibitory Interaction between the SH3 into the full-length Dock180 could functionally mimic the and Docker Domains of Dock180 effects of deleting the SH3 domain. Remarkably, the Previous studies showed a functional requirement for I32K mutation enhanced the activity of Dock180 in at least two regions of Dock180 during phagocytosis phagocytosis in a manner similar to that of the SH3- domain deletion (Figure 1C, lanes 7 and 8). When one *Correspondence: [email protected] considers that the I32K mutation disrupted the SH3 Current Biology 372 Figure 1. An Inhibitory Interaction between SH3 and Docker Domains of Dock180 (A) The PxxP motif of ELMO binds to the SH3 domain of Dock180. GST-Dock1-161 or its W45A or I32K mutant version was transfected into 293T cells with either full-length ELMO-GFP or its PxxP-deletion mutant. After GST precipitation, the bound proteins were analyzed by immunoblotting. (B) The second interaction between Dock180 and ELMO and its disruption by G171E mutation. Flag-Dock180 or one of its mutants was coexpressed with ELMO-GFP in 293T cells. Pre-cleared cell lysates were subject to anti-Flag precipitation. The bound proteins were analyzed by immunoblotting. The ELMO1-629, which does not bind Dock180 [1], was used as a negative control. (C) Phagocytosis assay. LR73 cells were transfected with wild-type Flag-Dock180 or one of its mutants together with either GFP (lanes 2–4) or ELMO-GFP (lanes 6–8). The percentage of GFP-positive cells with engulfed particles was determined in a flow-cytometry-based engulfment assay. The error bar represents the standard deviation. (D) The SH3 domain of Dock180 binds to the SH3 domain-deleted Dock180 mutant. Flag-Dock (⌬SH3) was coexpressed in 293T cells with either GST or GST-Dock1-161. Cleared cell lysates were precipitated with anti-Flag and immunoblotted with the indicated antibodies. (E) Interaction between the SH3 domain and the Docker domain of Dock180. Wild-type or mutant Flag-Docker domain and the indicated GST- Dock1-161 or its W45A mutant were expressed in 293T cells, precipitated with anti-Flag antibody, and immunoblotted with the indicated antibodies. The PP→AA mutation disrupted the single PxxP motif within the Docker domain. (F) I32K mutation within the SH3 domain disrupts SH3 interaction with the Docker domain. Flag-Docker domain was cotransfected with GST- Dock1-161 or its I32K mutant into 293T cells, precipitated with anti-Flag antibody, and immunoblotted with the indicated antibodies. Regulation of Dock180-Family GEFs 373 binding to Docker, these data support the hypothesis disrupted its binding to both the Docker domain and that the SH3:Docker interaction inhibits the functional ELMO (Figures 1A and 1F). Collectively, these results activity of Dock180. suggest that the binding of the PxxP motif of ELMO to the SH3 domain of Dock180 can disrupt the SH3:Docker interaction. SH3 Domain Blocks Rac Access Consistent with the above observations, the binding to the Docker Domain of an ELMO fragment carrying the PxxP motif enhanced Previous studies have shown that the binding of nucleo- Rac binding to Dock180 (Figure 3C, lanes 2 and 5) to tide-free Rac to the Docker domain is critical for Rac an extent roughly equal to that observed with the Dock activation by the Dock180/ELMO complex [1]. We hy- (⌬SH3) mutant (lanes 1 and 5). In contrast, coexpression pothesized that the SH3 domain might exert its inhibitory of ELMO (lanes 3 and 6) did not affect the binding of effect by blocking the access of Rac to the Docker nucleotide-free Rac to the Dock (W45A) mutant. Of note, domain. Interestingly, the Dock (⌬SH3) mutant associ- here we used the W665A mutant of ELMO to avoid the ated with nucleotide-free Rac significantly better than ELMO PH-domain-mediated contribution to Rac binding wild-type Dock180 (Figure 2A). A similar result was seen [18]. Similarly, ELMO interaction with Dock2 contributed with another CDM family member, Dock2 [17], wherein to enhanced Rac binding (Figure 2B). Collectively, these deletion of the N-terminal region resulted in better Rac data suggested that the PxxP motif of ELMO binding to binding (Figure 2B). Moreover, addition of nucleotide- the SH3 domain of CDM proteins could relieve the SH3- free Rac inhibited the SH3 domain binding to the Docker domain-mediated steric hindrance. domain (Figure 2C, lanes 2 and 3), suggesting that the We next assessed the functional relevance of the Dock- SH3 domain and nucleotide-free Rac competitively bind SH3:ELMO-PxxP interaction. In a fluorescence-based GEF the Docker domain. Under the same conditions, the GDP assay, in which the association between Dock180 and bound Rac, which does not bind the Docker domain with ELMO was primarily mediated via the SH3:PxxP interac- high affinity, did not reduce the SH3:Docker interaction tion, the Dock180/ELMO complex showed 2-fold en- (Figure 2C, lanes 5 and 6).
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